Project Abstract Spontaneous canine osteosarcoma (OS) is a well-defined large animal model of human OS, exhibiting similar clinical presentation and molecular aberrations. Indeed, canine OS has historically been used to help develop novel limb spare techniques, evaluate a variety of non-specific immunotherapeutics such as L-MTP-PE, and assess the efficacy of targeted therapeutics to prevent the outgrowth of metastatic disease. Despite best efforts, progress in the prevention and treatment of metastatic disease has essentially stalled for the past 3 decades; 30% of people and 90% of dogs still die of tumor spread, primarily to the lungs. Numerous clinical trials have been undertaken in people with macroscopic metastases and in dogs with both microscopic and macroscopic disease, yet all have failed to demonstrate improved outcomes. This is particularly evident with respect to immune checkpoint inhibitors that do not induce the dramatic disease regressions typically observed in other cancers such as melanoma and lung cancer. One particularly daunting challenge for immunotherapy-based therapeutics in OS relates to the permissiveness of the tumor microenvironment (TME) for inducing anti-tumor immune responses. Our data suggests that a relatively low mutational load combined with a dampened overall immune response in OS may contribute to the observed lack of response to current treatment approaches. We propose that targeting the immune suppressive TME in OS is essential to generating potent and durable anti- tumor immunity. To accomplish this, it may be necessary to simultaneously modulate several elements in the TME, including Tregs, MDSCs, M2 macrophages and overexpressed inhibitory checkpoint molecules. In support of this, we have generated a body of data demonstrating immunological activity of multiple therapeutics, including repurposed drugs with good safety records (losartan, oclacitinib), small molecule inhibitors with established PK/PD in dogs (toceranib, RV1001, reparixin, JHU-292) and antibodies specific for checkpoint molecules (anti- PD1). However, the exact combinations that are most effective against metastatic OS have not yet been identified, and this is a major goal of this proposal. As such, we hypothesize that an adaptive pilot trial design can be used to rapidly screen TME-targeting immunotherapy drug combinations in dogs with macroscopic chemotherapy-resistant metastatic OS and that this information can be refined to assess activity of the most active approach against microscopic metastases in a subsequent adjuvant trial. This will be accomplished by testing four TME modifying immunotherapy combinations for anti-tumor and immune modulatory activity in dogs with macroscopic OS metastases, interrogating relevant biomarkers associated with responses to therapy, then using this information to conduct an adjuvant immunotherapy trial with the most active combination in dogs with microscopic metastatic OS. The data generated from this proposal will create a blueprint for future immunotherapy studies in people with OS by eliminating approaches deemed inactive and generating a set of clinical biomarkers to guide treatment.